Molecular Identification of Indian Crocodile Species: PCR‐RFLP Method for Forensic Authentication*

Abstract

South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile-based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA-based identification of species using PCR-RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species-specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.

Complete mitochondrial genome sequences of three Crocodylus species and their comparison within the Order Crocodylia

“…Besides the evolutionary perspective, the polymorphisms in the mitochondrial genomes have been utilized for various ecological studies (Avise, 1995;Valentini et al, 2009). The forensic utility is another aspect where the mitochondrial genome sequences have proved useful for species identification and thereby are a valuable tool for the conservation of endangered species (Teletchea et al, 2005;Dubey et al, 2009Dubey et al, , 2010Meganathan et al, 2009aMeganathan et al, , 2010a. Thus, the generation of mitochondrial genome sequences is useful for phylogenetic analyses and also for informing conservation efforts.…”

Double gene targeting multiplex PCR-RFLP detects Crocodylus porosus in chicken meatball and traditional medicine

“…In other words, this novel mPCR-RFLP assay was sensitive, reliable, and robust for the discriminatory detection of crocodile in processed foods. [19] has carried out PCR-RFLP to discriminate C. porosus, C. palustris and C. gangeticus in fresh blood samples, nonetheless, the assay was not tested under food processing conditions. Previously, successful PCR-RFLP assay has been carried out in various food products, namely frankfurter, [27,42] meatballs, [34] burgers [29,41,43] for the detection of rabbit, rat, squirrel, beef, buffalo, pig, cat, dog, and monkey.…”

“…As authentication is concerned, six PCR assays [17][18][19][20][21][22] have been proposed for crocodile identification. Two of which are conventional PCR, [18,22] one PCR-RFLP [19] and the other three are multiplex PCR assays. [17,20,21] However, the documented methods are based on very long amplicon-lengths (373 bp-2000 bp) that are not so stable under extensive processing of food, pharmaceuticals, and cosmetics.…”

Validation of a Multiplex PCR Assay for the Forensic Identification of Indian Crocodiles*

“…Although law prevents any illegal activity against these species, illicit trade continues to date and wildlife forensics require effective molecular techniques for flawless identification of confiscated materials to prosecute the criminals. The existing molecular techniques like DNA sequencing (4), sequence characterized amplified region analysis (5), and polymerase chain reaction (PCR)–restriction fragment length polymorphism method (6) are time‐consuming and may not be efficient in case of mixed samples. Therefore, a simple multiplex PCR assay was developed based on cytochrome b (cyt b ) gene, in which the species‐specific forward primers: MUG for C. palustris , SAL for C. porosus , GHA for G. gangeticus and a universal reverse primer, UNI were designed to yield specific‐sized PCR products for each species.…”